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Abstract:

In an embodiment, the invention relates to the seeds, plants, and plant
parts of canola line ND-662c and to methods for producing a canola plant
produced by crossing canola line ND-662c with itself or with another
canola line. The invention also relates to methods for producing a canola
plant containing in its genetic material one or more transgenes and to
the transgenic canola plants and plant parts produced by those methods.
This invention also relates to canola lines or breeding lines and plant
parts derived from canola line ND-662c, to methods for producing other
canola lines, lines or plant parts derived from canola line ND-662c and
to the canola plants, varieties, and their parts derived from use of
those methods. The invention further relates to hybrid canola seeds,
plants and plant parts produced by crossing the line ND-662c with another
canola line.

Claims:

1. A seed of canola line ND-662c, representative sample of seed of which
was deposited under ATCC Accession No. PTA-11594.

2. A canola plant, or a part thereof, produced by growing the seed of
claim 1.

3. A tissue culture produced from protoplasts or cells from the plant of
claim 2, wherein said cells or protoplasts of the tissue culture are
produced from a plant part selected from the group consisting of leaf,
pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip,
anther, pistil, flower, shoot, stem, petiole and pod.

4. A canola plant regenerated from the tissue culture of claim 3, wherein
the plant has essentially all of the morphological and physiological
characteristics of line ND-662c as shown in Table 1.

5. A composition comprising a seed or plant part of canola line ND-662c
and a cultivation medium, wherein a representative sample of seed of
canola line ND-662c has been deposited under ATCC Accession No.
PTA-11594.

6. The composition of claim 5, wherein the cultivation medium is soil or
a synthetic medium.

7. A canola seed produced by crossing two canola plants and harvesting
the resultant canola seed, wherein at least one canola plant is the
canola plant of claim 2.

8. A canola plant, or a part thereof, produced by growing said seed of
claim 7.

9. A method of producing a male sterile canola plant wherein the method
comprises crossing the canola plant of claim 2 with a male sterile canola
plant and harvesting the resultant seed.

10. A male sterile canola plant produced by transforming the canola plant
of claim 2 with a nucleic acid molecule that confers male sterility.

14. A disease resistant canola plant produced by transforming the canola
plant of claim 2 with a transgene that confers disease resistance.

15. A canola plant having modified fatty acid metabolism or modified
carbohydrate metabolism produced by transforming the canola plant of
claim 2 with a transgene encoding a protein selected from the group
consisting of fructosyltransferase, levansucrase, alpha-amylase,
invertase and starch branching enzyme or encoding an antisense of
stearyl-ACP desaturase.

17. A method of introducing a desired trait into canola line ND-662c
wherein the method comprises: (a) crossing a ND-662c plant, wherein a
representative sample of seed was deposited under ATCC Accession No.
PTA-11594, with a plant of another canola line that comprises a desired
trait to produce progeny plants, wherein the desired trait is selected
from the group consisting of male sterility, herbicide resistance, insect
resistance, pest resistance, modified fatty acid metabolism, modified
carbohydrate metabolism, modified seed yield, modified oil percent,
modified protein percent, modified lodging resistance and resistance to
bacterial disease, fungal disease or viral disease; (b) selecting one or
more progeny plants that have the desired trait to produce selected
progeny plants; (c) crossing the selected progeny plants with the ND-662c
plants to produce backcross progeny plants; (d) selecting for backcross
progeny plants that have the desired trait and essentially all of the
physiological and morphological characteristics of canola line ND-662c
listed in Table 1; and (e) repeating steps (c) and (d) two or more times
to produce selected third or higher backcross progeny plants that
comprise the desired trait and essentially all of the physiological and
morphological characteristics of canola line ND-662c as shown in Table 1.

18. A canola plant produced by the method of claim 17, wherein the plant
has the desired trait.

20. The canola plant of claim 18, wherein the desired trait is insect
resistance and the insect resistance is conferred by a transgene encoding
a Bacillus thuringiensis endotoxin.

21. The canola plant of claim 18, wherein the desired trait is modified
fatty acid metabolism or modified carbohydrate metabolism and said
desired trait is conferred by a nucleic acid encoding a protein selected
from the group consisting of phytase, fructosyltransferase, levansucrase,
α-amylase, invertase and starch branching enzyme or encoding an
antisense of stearyl-ACP desaturase.

Description:

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a new and distinctive canola line,
designated ND-662c. All publications cited in this application are herein
incorporated by reference.

[0003] 2. Description of Related Art

[0004] Canola, Brassica napus oleifera annua, is an important and valuable
field crop. Thus, a continuing goal of canola plant breeders is to
develop stable, high yielding canola lines that are agronomically sound.
The reasons for this goal are obviously to maximize the amount of grain
produced on the land used and to supply food for both animals and humans.
The high quality vegetable oil extracted from canola grain is a primary
reason for canola's commercial value. Thus, in addition to breeding
varieties that offer high grain yields, canola plant breeders also focus
on increasing the oil content level in the grain in order to maximize
total oil yield per acre. To accomplish these goals, the canola breeder
must select and develop canola plants that have the traits that result in
superior lines.

SUMMARY OF THE INVENTION

[0005] Reference now will be made in detail to the embodiments of the
invention, one or more examples of which are set forth below. Each
example is provided by way of explanation of the invention, not a
limitation of the invention. In fact, it will be apparent to those
skilled in the art that various modifications and variations can be made
in the present invention without departing from the scope or spirit of
the invention. For instance, features illustrated or described as part of
one embodiment, can be used on another embodiment to yield a still
further embodiment.

[0006] Thus, it is intended that the present invention covers such
modifications and variations as come within the scope of the appended
claims and their equivalents. Other objects, features and aspects of the
present invention are disclosed in or are obvious from the following
detailed description. It is to be understood by one of ordinary skill in
the art that the present discussion is a description of exemplary
embodiments only, and is not intended as limiting the broader aspects of
the present invention.

[0007] According to the invention, there is provided a new canola line
designated ND-662c. This invention thus relates to the seeds, plants,
and/or plant parts of canola of canola line ND-662c and to methods for
producing a canola plant produced by crossing the canola ND-662c with
itself or another canola genotype, and the creation of variants by
mutagenesis or transformation of canola ND-662c.

[0008] Thus, any methods using the canola line ND-662c are part of this
invention: selfing, backcrosses, hybrid production, crosses to
populations, and the like. All plants produced using canola line ND-662c
as a parent are within the scope of this invention. Advantageously, the
canola line could be used in crosses with other, different, canola plants
to produce first generation (F1) canola hybrid seeds and plants with
superior characteristics.

[0010] In another aspect, the present invention provides regenerable cells
for use in tissue culture of canola plant ND-662c. The tissue culture
will preferably be capable of regenerating plants having essentially all
of the physiological and morphological characteristics of the foregoing
canola plant, and of regenerating plants having substantially the same
genotype as the foregoing canola plant. Preferably, the regenerable cells
in such tissue cultures will be embryos, protoplasts, meristematic cells,
callus, pollen, leaves, anthers, pistils, cotyledons, roots, root tips,
flowers, seeds, pods or stems. Still further, the present invention
provides canola plants regenerated from the tissue cultures of the
invention.

[0011] In another aspect, the present invention provides a method of
introducing a desired trait into canola line ND-662c wherein the method
comprises: (1) crossing a ND-662c plant with a plant of another canola
genotype that comprises a desired trait to produce F1 progeny
plants, wherein the desired trait is selected from the group consisting
of male sterility, herbicide resistance, insect resistance, modified
fatty acid metabolism, modified carbohydrate metabolism, modified seed
yield, modified oil percent, modified protein percent, modified lodging
resistance and resistance to bacterial disease, fungal disease or viral
disease; (2) selecting one or more progeny plants that have the desired
trait to produce selected progeny plants; (3) crossing the selected
progeny plants with the ND-662c plants to produce backcross progeny
plants; (4) selecting for backcross progeny plants that have the desired
trait and essentially all of the physiological and morphological
characteristics of canola line ND-662c to produce selected backcross
progeny plants; and (5) repeating these steps three or more times to
produce selected fourth or higher backcross progeny plants that comprise
the desired trait and essentially all of the physiological and
morphological characteristics of canola line ND-662c as listed in Table
1. Included in this aspect of the invention is the plant produced by the
method wherein the plant has the desired trait and essentially all of the
physiological and morphological characteristics of canola line ND-662c as
listed in Table 1.

DEFINITIONS

[0012] In the description and tables which follow, a number of terms are
used. In order to provide a clear and consistent understanding of the
specification and claims, including the scope to be given such terms, the
following definitions are provided:

[0013] Allele. Allele is any of one or more alternative forms of a gene
which relate to one trait or characteristic. In a diploid cell or
organism, the two alleles of a given gene occupy corresponding loci on a
pair of homologous chromosomes.

[0014] Alter. The utilization of up-regulation, down-regulation, or gene
silencing.

[0015] Anther arrangement. The orientation of the anthers in fully opened
flowers can also be useful as an identifying trait. This can range from
introse (facing inward toward pistil), erect (neither inward not
outward), or extrose (facing outward away from pistil).

[0016] Anther dotting. The presence/absence of anther dotting (colored
spots on the tips of anthers) and if present, the percentage of anther
dotting on the tips of anthers in newly opened flowers is also a
distinguishing trait for varieties.

[0017] Anther fertility. This is a measure of the amount of pollen
produced on the anthers of a flower. It can range from sterile (such as
in female parents used for hybrid seed production) to fertile (all
anthers shedding).

[0018] Backcrossing. Backcrossing is a process in which a breeder
repeatedly crosses hybrid progeny back to one of the parents, for
example, a first generation hybrid F1 with one of the parental
genotypes of the F1 hybrid.

[0019] Blackleg (Leptosphaeria maculans). Virulent or severe blackleg of
canola/rapeseed is a fungal canker or dry rot disease of the actively
growing crop that causes stem girdling and lodging. In heavily infested
crops, up to 100 percent of the stems may be infected, resulting in major
yield loss. For purposes of this application, resistance to blackleg is
measured using ratings of "R" (resistant), "MR" (medium resistant), "MS"
(moderately susceptible) or "S" (susceptible).

[0020] Cell. Cell as used herein includes a plant cell, whether isolated,
in tissue culture or incorporated in a plant or plant part.

[0021] Cotyledon width. The cotyledons are leaf structures that form in
the developing seeds of canola which make up the majority of the mature
seed of these species. When the seed germinates, the cotyledons are
pushed out of the soil by the growing hypocotyls (segment of the seedling
stem below the cotyledons and above the root) and they unfold as the
first photosynthetic leafs of the plant. The width of the cotyledons
varies by variety and can be classified as narrow, medium, or wide.

[0022] Early vigor. This is a measure of a canola seed's ability to
germinate and emerge after planting. A score of 1 indicates poor early
vigor. A score of 5 indicates excellent early vigor. The early vigor
rating is taken at four weeks after planting.

[0023] Elite canola line. A canola line, per se, which has been sold
commercially.

[0024] Elite canola parent line. A canola line which is the parent line of
a canola hybrid which has been commercially sold.

[0025] Embryo. The embryo is the small plant contained within a mature
seed.

[0026] Essentially all of the physiological and morphological
characteristics. "Essentially all of the physiological and morphological
characteristics" refers to a plant having essentially all of the
physiological and morphological characteristics of the recurrent parent,
except for the characteristics derived from the converted trait.

[0027] FAME analysis. Fatty Acid Methyl Ester analysis is a method that
allows for accurate quantification of the fatty acids that make up
complex lipid classes.

[0028] Flower bud location. The location of the unopened flower buds
relative to the adjacent opened flowers is useful in distinguishing
between the canola species. The unopened buds are held above the most
recently opened flowers in B. napus and they are positioned below the
most recently opened flower buds in B. rapa.

[0029] Flowering date. This is measured by the number of days from
planting to the stage when 50% of the plants in a population have one or
more open flowers. This varies from variety to variety.

[0030] Fusarium Wilt. Fusarium wilt, largely caused by Fusarium oxysporum,
is a disease of canola that causes part or all of a plant to wilt,
reducing yield by up to 30% or more on badly affected fields. For
purposes of this application, resistance to Fusarium wilt is measured
using ratings of "R" (resistant), "MR" (medium resistant), "MS"
(moderately susceptible) or "S" (susceptible).

[0031] Gene silencing. Gene silencing means the interruption or
suppression of the expression of a gene at the level of transcription or
translation.

[0032] Genotype. Refers to the genetic constitution of a cell or organism.
Glucosinolates. These are measured in micromoles (μm) of total
alipathic glucosinolates per gram of air-dried oil-free meal. The level
of glucosinolates is somewhat influenced by the sulfur fertility of the
soil, but is also controlled by the genetic makeup of each variety and
thus can be useful in characterizing varieties.

[0033] Growth habit. At the end of flowering, the angle relative to the
ground surface of the outermost fully expanded leaf petioles is a variety
specific trait. This trait can range from erect (very upright along the
stem) to prostrate (almost horizontal and parallel with the ground
surface).

[0034] Leaf attachment to the stem. This trait is especially useful for
distinguishing between the two canola species. The base of the leaf blade
of the upper stem leaves of B. rapa completely clasp the stem whereas
those of the B. napus only partially clasp the stem. Those of the mustard
species do not clasp the stem at all.

[0035] Leaf blade color. The color of the leaf blades is variety specific
and can range from light to medium dark green to blue green.

[0036] Leaf development of lobes. The leaves on the upper portion of the
stem can show varying degrees of development of lobes which are
disconnected from one another along the petiole of the leaf. The degree
of lobing is variety specific and can range from absent (no lobes)/weak
through very strong (abundant lobes).

[0037] Leaf glaucosity. This refers to the waxiness of the leaves and is
characteristic of specific varieties although environment can have some
effect on the degree of waxiness. This trait can range from absent (no
waxiness)/weak through very strong. The degree of waxiness can be best
determined by rubbing the leaf surface and noting the degree of wax
present.

[0038] Leaf indentation of margin. The leaves on the upper portion of the
stem can also show varying degrees of serration along the leaf margins.
The degree of serration or indentation of the leaf margins can vary from
absent (smooth margin)/weak to strong (heavy saw-tooth like margin).

[0039] Leaf pubescence. The leaf pubescence is the degree of hairiness of
the leaf surface and is especially useful for distinguishing between the
canola species. There are two main classes of pubescence which are
glabrous (smooth/not hairy) and pubescent (hairy) which mainly
differentiate between the B. napus and B. rapa species, respectively.

[0040] Leaf surface. The leaf surface can also be used to distinguish
between varieties. The surface can be smooth or rugose (lumpy) with
varying degrees between the two extremes.

[0041] Linkage. Refers to a phenomenon wherein alleles on the same
chromosome tend to segregate together more often than expected by chance
if their transmission was independent.

[0042] Linkage disequilibrium. Refers to a phenomenon wherein alleles tend
to remain together in linkage groups when segregating from parents to
offspring, with a greater frequency than expected from their individual
frequencies.

[0043] Locus. A locus confers one or more traits such as, for example,
male sterility, herbicide tolerance, insect resistance, disease
resistance, modified fatty acid metabolism, modified phytic acid
metabolism, modified carbohydrate metabolism and modified protein
metabolism. The trait may be, for example, conferred by a naturally
occurring gene introduced into the genome of the variety by backcrossing,
a natural or induced mutation, or a transgene introduced through genetic
transformation techniques. A locus may comprise one or more alleles
integrated at a single chromosomal location.

[0044] Lodging resistance. Lodging is rated on a scale of 0 to 5. A score
of 0 indicates plants are lying on the ground (lodged). A score of 5
indicates plants are standing vertical (not lodged).

[0045] Maturity. The maturity of a variety is measured as the number of
days between planting and physiological maturity. This is useful trait in
distinguishing varieties relative to one another.

[0046] Oil content. This is measured as percent of the whole dried seed
and is characteristic of different varieties. It can be determined using
various analytical techniques such as NMR, NIR, and Soxhlet extraction.

[0047] Open pollination. As used herein, the term open pollination means
the natural, uncontrolled pollination, such as by insects, birds, wind,
or other natural mechanisms, that occurs within a cross-pollinated or
self-pollinated population or variety.

[0048] Percent linolenic acid. Percent oil of the seed that is linolenic
acid.

[0050] Percentage of total fatty acids. This is determined by extracting a
sample of oil from seed, producing the methyl esters of fatty acids
present in that oil sample and analyzing the proportions of the various
fatty acids in the sample using gas chromatography. The fatty acid
composition can also be a distinguishing characteristic of a variety.

[0051] Petal color. The petal color on the first day a flower opens can be
a distinguishing characteristic for a variety. It can be white, varying
shades of yellow or orange.

[0052] Plant. As used herein, the term "plant" includes reference to an
immature or mature whole plant, including a plant from which seed or
grain or anthers have been removed. Seed or embryo that will produce the
plant is also considered to be the plant.

[0053] Plant height. This is the height of the plant at the end of
flowering if the floral branches are extended upright (i.e., not lodged).
This varies from variety to variety and although it can be influenced by
environment, relative comparisons between varieties grown side by side
are useful for variety identification.

[0055] Protein content. This is measured as percent of whole dried seed
and is characteristic of different varieties. This can be determined
using various analytical techniques such as NIR and Kjeldahl.

[0056] Quantitative trait loci (QTL). Quantitative trait loci (QTL) refer
to genetic loci that control to some degree numerically representable
traits that are usually continuously distributed.

[0057] Regeneration. Regeneration refers to the development of a plant
from tissue culture.

[0058] Resistance to lodging. This measures the ability of a variety to
stand up in the field under high yield conditions and severe
environmental factors. A variety can have good (remains upright), fair,
or poor (falls over) resistance to lodging. The degree of resistance to
lodging is not expressed under all conditions but is most meaningful when
there is some degree of lodging in a field trial.

[0059] Sclerotinia. The fungal pathogen Sclerotinia sclerotiorum causes
Sclerotinia, also called white mold, in various crop species including
canola. In canola, symptoms first appear in the flowers and then, as
conditions are favorable, spread to the branches and stems. If infection
occurs in the main stem (Sclerotinia Stem Rot or SSR), the plants may die
early and become prone to lodging. In heavily infested crops, 50 percent
or more of the stems may be infected, resulting in major yield loss. For
purposes of this application, resistance to blackleg is measured using
ratings of "R" (resistant), "MR" (medium resistant), "MS" (moderately
susceptible) or "S" (susceptible).

[0060] Seed coat color. The color of the seed coat can be variety specific
and can range from black through brown through yellow. Color can also be
mixed for some varieties.

[0061] Seed coat mucilage. This is useful for differentiating between the
two species of canola with B. rapa varieties having mucilage present in
their seed coats whereas B. napus varieties do not have this present. It
is detected by imbibing seeds with water and monitoring the mucilage that
is exuded by the seed.

[0062] Seedling growth habit. The rosette consists of the first 2-8 true
leaves and a variety can be characterized as having a strong rosette
(closely packed leaves) or a weak rosette (loosely arranged leaves).

[0063] Silique (pod) habit. This is also a trait which is variety specific
and is a measure of the orientation of the pods along the racemes
(flowering stems). This trait can range from erect (pods angled close to
racemes) through horizontal (pods perpendicular to racemes) through
arching (pods show distinct arching habit).

[0064] Silique (pod) length of beak. The beak is the segment at the end of
the pod which does not contain seed (it is a remnant of the stigma and
style for the flower). The length of the beak can be variety specific and
can range form short through medium through long.

[0065] Silique (pod) length of pedicel. The pedicel is the stem that
attaches the pod to the raceme of flowering shoot. The length of the
pedicel can be variety specific and can vary from short through medium
through long.

[0066] Silique (pod) length. This is the length of the fully developed
pods and can range from short to medium to long. It is best used by
making comparisons relative to reference varieties.

[0067] Silique (pod) type. This is typically a bilateral single pod for
both species of canola and is not really useful for variety
identification within these species.

[0068] Silique (pod) width. This is the width of the fully developed pods
and can range from narrow to medium to wide. It is best used by making
comparisons relative to reference varieties.

[0069] Single gene converted (conversion). Single gene converted
(conversion) plant refers to plants which are developed by a plant
breeding technique called backcrossing, or via genetic engineering,
wherein essentially all of the desired morphological and physiological
characteristics of a variety are recovered in addition to the single gene
transferred into the variety via the backcrossing technique or via
genetic engineering.

[0070] Stem intensity of anthocyanin coloration. The stems and other
organs of canola plants can have varying degrees of purple coloration
which is due to the presence of anthocyanin (purple) pigments. The degree
of coloration is somewhat subject to growing conditions, but varieties
typically show varying degrees of coloration ranging from: absent (no
purple)/very weak to very strong (deep purple coloration).

[0071] Total saturated (TOTSAT). Total percent oil of the seed of the
saturated fats in the oil including C12:0, C14:0, C16:0, C18:0, C20:0,
C22:0 and C24.0.

DETAILED DESCRIPTION OF THE INVENTION

[0072] Spring canola variety ND-662c is a spring canola variety developed
from the initial cross, made in 1998, of SCV574782 (a proprietary
Roundup®-tolerant spring canola line of Monsanto Technology LLC) and
46A65 (a commercial, open pollinated canola variety developed and sold by
Pioneer Hi-Bred Production Limited, a subsidiary of Pioneer Hi-Bred
International). Progeny from this cross were self-pollinated and the
pedigree system of plant breeding was then used to develop ND-662c. Some
of the criteria used for selection in various generations include:
ROUNDUP READY® trait, standability, oil content, maturity, and
resistance to blackleg disease (Leptosphaeria maculans).

[0073] At the F7 generation, the line was coded PR6377 for testing in
the first year of Canadian registration trials in 2001. It was again
tested in 2002, before being advanced to Canadian public registration
trials in 2003. Individual plants with improved blackleg resistance were
increased and grown in Chile during the 2002/2003 contra season to
produce the seed tested in the 2003 registration trials. The line was
then re-selected for individuals with lower total saturated fatty acids
in the seed oil. These individuals were increased in bulk and recoded at
the F11 generation as ND-662c.

[0074] 46A65, the original male parent line, was first made available for
sale in Canada about 1996, the year in which an application for Plant
Breeders Rights was submitted by Pioneer Hi-Bred in Canada (application
withdrawn in 1999). In addition, Pioneer Hi-Bred International filed a
U.S. PVP application (#9800047) for 46A65 on Dec. 22, 1997, which was
abandoned on Jun. 20, 2006.

[0075] ND-662c is a ROUNDUP READY® pollen fertile spring canola variety
with above average oil content, resistance to blackleg disease and medium
resistance to sclerotinium stem rot. Canola line ND-662c is stable,
uniform, and no off-type plants have been exhibited in evaluation. It
meets all canola quality standards.

[0076] The line has shown uniformity and stability, as described in the
following variety description information. The line has been increased
with continued observation for uniformity. Variants, visible as slightly
taller plants, may occur at a frequency of less than 0.1%.

[0077] Compared to two commercial check varieties, DKL38-25 and IS 71-45,
canola line ND-662c has the following morphologic and other
characteristics.

[0078] Compared to the average of the values measured for DKL38-25 and IS
71-45, ND-662c has a days to maturity rating that is more than 1.5 days
earlier, a shorter plant height, a comparable lodging resistance rating
that is within a normal range for commercial canola, comparable
resistance to blackleg and Sclerotinia stem rot, a higher seed yield per
acre, a higher total oil yield, a higher oil content, a lower protein
content, and similarly low erucic acid and glucosinolate contents that
are within acceptable ranges for canola.

[0079] This invention is also directed to methods for producing a canola
plant by crossing a first parent canola plant with a second parent canola
plant, wherein the first or second canola plant is the canola plant from
the line ND-662c. Further, both first and second parent canola plants may
be from the line ND-662c. Therefore, any methods using the line ND-662c
are part of this invention: selling, backcrosses, hybrid breeding, and
crosses to populations. Any plants produced using line ND-662c as a
parent are within the scope of this invention.

[0080] Additional methods using the ND-662c line include, but are not
limited to, expression vectors introduced into plant tissues using a
direct gene transfer method such as microprojectile-mediated delivery,
DNA injection, electroporation, and the like. More preferably, expression
vectors may be introduced into plant tissues by using either
microprojectile-mediated delivery with a ballistic device or by using
Agrobacterium-mediated transformation. Transformant plants obtained with
the protoplasm of the invention are intended to be within the scope of
this invention.

[0081] The advent of new molecular biological techniques has allowed the
isolation and characterization of genetic elements with specific
functions, such as encoding specific protein products. Scientists in the
field of plant biology developed a strong interest in engineering the
genome of plants to contain and express foreign genetic elements, or
additional, or modified versions of native or endogenous genetic elements
in order to alter the traits of a plant in a specific manner. Any DNA
sequences, whether from a different species or from the same species
which are inserted into the genome using transformation, are referred to
herein collectively as "transgenes." In some embodiments of the
invention, a transgenic variant of ND-662c may contain at least one
transgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or
no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. Over the
last fifteen to twenty years, several methods for producing transgenic
plants have been developed, and the present invention also relates to
transgenic variants of the claimed canola line ND-662c.

[0082] One embodiment of the invention is a process for producing canola
line ND-662c further comprising a desired trait, said process comprising
transforming a canola plant of line ND-662c with a transgene that confers
a desired trait. Another embodiment is the product produced by this
process. In one embodiment the desired trait may be one or more of
herbicide resistance, insect resistance, disease resistance, modified
seed yield, modified oil percent, modified protein percent, modified
lodging resistance or modified fatty acid or carbohydrate metabolism. The
specific gene may be any known in the art or listed herein, including; a
polynucleotide conferring resistance to imidazolinone, sulfonylurea,
glyphosate, glufosinate, triazine, hydroxyphenylpyruvate dioxygenase
inhibitor, protoporphyrinogen oxidase inhibitor and benzonitrile; a
polynucleotide encoding a Bacillus thuringiensis polypeptide, a
polynucleotide encoding phytase, FAD-2, FAD-3, galactinol synthase or a
raffinose synthetic enzyme; or a polynucleotide conferring resistance to
blackleg, white rust or other common canola diseases.

[0083] Numerous methods for plant transformation have been developed,
including biological and physical plant transformation protocols, all of
which may be used with this invention. In addition, expression vectors
and in vitro culture methods for plant cell or tissue transformation and
regeneration of plants are available and may be used in conjunction with
the invention.

[0084] In an embodiment, a genetic trait which has been engineered into
the genome of a particular canola plant may be moved into the genome of
another variety, such as canola line ND-662c, using traditional breeding
techniques that are well known in the plant breeding arts. For example, a
backcrossing approach may be used to move a transgene from a transformed
canola variety into an already developed canola variety, and the
resulting backcross conversion plant would then comprise the
transgene(s).

[0085] In embodiments, various genetic elements can be introduced into the
plant genome using transformation. These elements include any known in
the art, specifically including, but not limited to genes, coding
sequences, inducible, constitutive, and tissue specific promoters,
enhancing sequences, and signal and targeting sequences.

[0086] Plant transformation involves the construction of an expression
vector which will function in plant cells. Such a vector comprises DNA
comprising a gene under control of or operatively linked to a regulatory
element (for example, a promoter). The expression vector may contain one
or more of such operably linked gene/regulatory element combinations. The
vector(s) may be in the form of a plasmid, and can be used alone or in
combination with other plasmids, to provide transformed canola plants,
using transformation methods as described below to incorporate transgenes
into the genetic material of the canola plant(s).

Expression Vectors for Canola Transformation: Marker Genes

[0087] Expression vectors include at least one genetic marker operably
linked to a regulatory element (a promoter, for example) that allows
transformed cells containing the marker to be either recovered by
negative selection, i.e., inhibiting growth of cells that do not contain
the selectable marker gene, or by positive selection, i.e., screening for
the product encoded by the genetic marker. Many commonly used selectable
marker genes for plant transformation are well known in the
transformation arts, and include, for example, genes that code for
enzymes that metabolically detoxify a selective chemical agent which may
be an antibiotic or an herbicide, or genes that encode an altered target
which is insensitive to the inhibitor. A few positive selection methods
are also known in the art.

[0088] One commonly used selectable marker gene for plant transformation
is the neomycin phosphotransferase II (nptII) gene which, when under the
control of plant regulatory signals, confers resistance to kanamycin.
Another commonly used selectable marker gene is the hygromycin
phosphotransferase gene which confers resistance to the antibiotic
hygromycin.

[0090] Another class of marker genes for plant transformation requires
screening of presumptively transformed plant cells rather than direct
genetic selection of transformed cells for resistance to a toxic
substance such as an antibiotic. These genes are particularly useful to
quantify or visualize the spatial pattern of expression of a gene in
specific tissues and are frequently referred to as reporter genes because
they can be fused to a gene or gene regulatory sequence for the
investigation of gene expression. Commonly used genes for screening
presumptively transformed cells include β-glucuronidase (GUS),
β-galactosidase, luciferase and chloramphenicol acetyltransferase.
Any of the above, or other marker genes, may be utilized in the present
invention.

[0091] In vivo methods for visualizing GUS activity that do not require
destruction of plant tissue are available and can be used in embodiments
of the invention. Additionally, Green Fluorescent Protein (GFP) can be
utilized as a marker for gene expression in prokaryotic and eukaryotic
cells. GFP and mutants of GFP may be used as screenable markers.

Expression Vectors for Canola Transformation: Promoters

[0092] Genes included in expression vectors must be driven by a nucleotide
sequence comprising a regulatory element, for example, a promoter.
Several types of promoters are well known in the transformation arts, as
are other regulatory elements that can be used alone or in combination
with promoters.

[0093] As used herein, "promoter" includes reference to a region of DNA
upstream from the start of transcription and involved in recognition and
binding of RNA polymerase and other proteins to initiate transcription. A
"plant promoter" is a promoter capable of initiating transcription in
plant cells. Examples of promoters under developmental control include
promoters that preferentially initiate transcription in certain tissues,
such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or
sclerenchyma. Such promoters are referred to as "tissue-preferred".
Promoters which initiate transcription only in certain tissues are
referred to as "tissue-specific". A "cell type" specific promoter
primarily drives expression in certain cell types in one or more organs,
for example, vascular cells in roots or leaves. An "inducible" promoter
is a promoter which is under environmental control. Examples of
environmental conditions that may effect transcription by inducible
promoters include anaerobic conditions or the presence of light.
Tissue-specific, tissue-preferred, cell type specific, and inducible
promoters constitute the class of "non-constitutive" promoters. A
"constitutive" promoter is a promoter which is active under most
environmental conditions.

[0094] A. Inducible Promoters--An inducible promoter is operably linked to
a gene for expression in canola. Optionally, the inducible promoter is
operably linked to a nucleotide sequence encoding a signal sequence which
is operably linked to a gene for expression in canola. With an inducible
promoter the rate of transcription increases in response to an inducing
agent.

[0095] Any inducible promoter can be used in the instant invention.
Exemplary inducible promoters include, but are not limited to, those from
the ACEI system which respond to copper, the In2 gene from maize which
responds to benzenesulfonamide herbicide safeners, or the Tet repressor
from Tn10. A particularly preferred inducible promoter is a promoter that
responds to an inducing agent to which plants do not normally respond. An
exemplary inducible promoter is the inducible promoter from a steroid
hormone gene, the transcriptional activity of which is induced by a
glucocorticosteroid hormone.

[0096] B. Constitutive Promoters--A constitutive promoter is operably
linked to a gene for expression in canola or the constitutive promoter is
operably linked to a nucleotide sequence encoding a signal sequence which
is operably linked to a gene for expression in canola.

[0097] Many different constitutive promoters can be utilized in the
instant invention. Exemplary constitutive promoters include, but are not
limited to, the promoters from plant viruses such as the 35S promoter
from CaMV and the promoters from such genes as rice actin, ubiquitin,
pEMU, MAS, and maize H3 histone. The ALS promoter, Xbal/Ncol fragment 5'
to the Brassica napus ALS3 structural gene (or a nucleotide sequence
similarity to said Xbal/Ncol fragment) could also be utilized herein.

[0098] C. Tissue-specific or Tissue-preferred Promoters--A tissue-specific
promoter is operably linked to a gene for expression in canola.
Optionally, the tissue-specific promoter is operably linked to a
nucleotide sequence encoding a signal sequence which is operably linked
to a gene for expression in canola. Plants transformed with a gene of
interest operably linked to a tissue-specific promoter produce the
protein product of the transgene exclusively, or preferentially, in a
specific tissue.

[0099] Any tissue-specific or tissue-preferred promoter can be utilized in
the instant invention. Exemplary tissue-specific or tissue-preferred
promoters include, but are not limited to, a root-preferred promoter such
as that from the phaseolin gene, a leaf-specific and light-induced
promoter such as that from cab or rubisco, an anther-specific promoter
such as that from LAT52, a pollen-specific promoter such as that from
Zm13, or a microspore-preferred promoter such as that from apg.

Signal Sequences for Targeting Proteins to Subcellular Compartments

[0100] Transport of protein produced by transgenes to a subcellular
compartment such as the chloroplast, vacuole, peroxisome, glyoxysome,
cell wall or mitochondrion or for secretion into the apoplast, is
accomplished by means of operably linking the nucleotide sequence
encoding a signal sequence to the 5' and/or 3' region of a gene encoding
the protein of interest. Targeting sequences at the 5' and/or 3' end of
the structural gene may determine, during protein synthesis and
processing, where the encoded protein is ultimately compartmentalized.
The presence of a signal sequence directs a polypeptide to either an
intracellular organelle or subcellular compartment or for secretion to
the apoplast. Many signal sequences are known in the art and can be
utilized in the present invention.

Foreign Protein Genes and Agronomic Genes

[0101] With transgenic plants according to the present invention, a
foreign protein can be produced in commercial quantities. Thus,
techniques for the selection and propagation of transformed plants, which
are well understood in the art, are within the scope of the invention. In
an embodiment, a foreign protein then can be extracted from a tissue of
interest or from the total biomass by known methods.

[0102] According to a preferred embodiment, the transgenic plant provided
for commercial production of foreign protein is a canola plant. In
another preferred embodiment, the biomass of interest is seed. For the
relatively small number of transgenic plants that show higher levels of
expression, a genetic map can be generated, primarily via conventional
RFLP, PCR and SSR analysis, which identifies the approximate chromosomal
location of the integrated DNA molecule. Map information concerning
chromosomal location is useful for proprietary protection of a subject
transgenic plant. If unauthorized propagation is undertaken and crosses
are made with other germplasm, the map of the integration region can be
compared to similar maps for suspect plants, to determine if the latter
have a common parentage with the subject plant. Map comparisons would
involve hybridizations, RFLP, PCR, SSR and sequencing, all of which are
conventional techniques. SNPs may also be used alone or in combination
with other techniques.

[0103] Likewise, by means of the present invention, plants can be
genetically engineered to express various phenotypes of agronomic
interest. Through the transformation of canola, the expression of genes
can be altered to enhance disease resistance, insect resistance,
herbicide resistance, agronomic, grain quality and other traits.
Transformation can also be used to insert DNA sequences which control, or
help control, male-sterility. DNA sequences native to canola, as well as
non-native DNA sequences, can be transformed into canola and used to
alter levels of native or non-native proteins. Various promoters,
targeting sequences, enhancing sequences, and other DNA sequences can be
inserted into the genome for the purpose of altering the expression of
proteins. Reduction of the activity of specific genes (also known as gene
silencing, or gene suppression) is desirable for several aspects of
genetic engineering in plants.

[0104] Many techniques for gene silencing are well known to one of skill
in the art, including but not limited to knock-outs (such as by insertion
of a transposable element such as Mu or other genetic elements such as a
FRT, Lox or other site specific integration site), antisense technology,
co-suppression, RNA interference, virus-induced gene silencing,
target-RNA-specific ribozymes, hairpin structures, MicroRNA, ribozymes,
oligonucleotide-mediated targeted modification, Zn-finger targeted
molecules, and other methods or combinations of the above methods known
to those of skill in the art.

[0105] Likewise, by means of the present invention, agronomic genes can be
expressed in transformed plants. More particularly, plants can be
genetically engineered to express various phenotypes of agronomic
interest. Exemplary genes implicated in this regard include, but are not
limited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:
[0106] A. Plant disease resistance genes. Plant defences are often
activated by specific interaction between the product of a disease
resistance gene (R) in the plant and the product of a corresponding
avirulence (Avr) gene in the pathogen. A plant variety can be transformed
with cloned resistance genes to engineer plants that are resistant to
specific pathogen strains. [0107] B. A gene conferring resistance to
fungal pathogens, such as oxalate oxidase or oxalate decarboxylase.
[0108] C. A Bacillus thuringiensis protein, a derivative thereof, or a
synthetic polypeptide modeled thereon, for example, a Bt
δ-endotoxin gene. [0109] D. A lectin. [0110] E. A vitamin-binding
protein such as avidin or a homolog. [0111] F. An enzyme inhibitor, for
example, a protease or proteinase inhibitor or an amylase inhibitor.
[0112] G. An insect-specific hormone or pheromone such as an ecdysteroid
or juvenile hormone, a variant thereof, a mimetic based thereon, or an
antagonist or agonist thereof. [0113] H. An insect-specific peptide or
neuropeptide which, upon expression, disrupts the physiology of the
affected pest. [0114] I. An insect-specific venom produced in nature by a
snake, a wasp, etc. [0115] J. An enzyme responsible for a
hyperaccumulation of a monoterpene, a sesquiterpene, a steroid,
hydroxamic acid, a phenylpropanoid derivative or another non-protein
molecule with insecticidal activity. [0116] K. An enzyme involved in the
modification, including the post-translational modification, of a
biologically active molecule; for example, a glycolytic enzyme, a
proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a
transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a
phosphorylase, a polymerase, an elastase, a chitinase and a glucanase,
whether natural or synthetic. [0117] L. A molecule that stimulates signal
transduction. [0118] M. A hydrophobic moment peptide. [0119] N. A
membrane permease, a channel former or a channel blocker. [0120] O. A
viral-invasive protein or a complex toxin derived therefrom. For example,
the accumulation of viral coat proteins in transformed plant cells
imparts resistance to viral infection and/or disease development effected
by the virus from which the coat protein gene is derived, as well as by
related viruses. Coat protein-mediated resistance has been conferred upon
transformed plants against alfalfa mosaic virus, cucumber mosaic virus,
tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus,
tobacco rattle virus and tobacco mosaic virus. [0121] P. An
insect-specific antibody or an immunotoxin derived therefrom. An antibody
targeted to a critical metabolic function in the insect gut would
inactivate an affected enzyme, killing the insect. [0122] Q. A
virus-specific antibody. [0123] R. A developmental-arrestive protein
produced in nature by a pathogen or a parasite. Thus, fungal
endo-α-1,4-D-polygalacturonases facilitate fungal colonization and
plant nutrient release by solubilizing plant cell wall
homo-α-1,4-D-galacturonase. [0124] S. A developmental-arrestive
protein produced in nature by a plant. [0125] T. Genes involved in the
Systemic Acquired Resistance (SAR) Response and/or the
pathogenesis-related genes. [0126] U. Antifungal genes. [0127] V.
Detoxification genes, such as for fumonisin, beauvericin, moniliformin
and zearalenone and their structurally related derivatives. [0128] W.
Cystatin and cysteine proteinase inhibitors. [0129] X. Defensin genes.
[0130] Y. Genes that confer resistance to Phytophthora root rot, such as
the Brassica equivalents of the Rps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps
1-d, Rps 1-e, Rps 1-k, Rps 2, Rps 3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5,
Rps 6, Rps 7 and other Rps genes. 2. Genes that Confer Resistance to an
Herbicide, for Example: [0131] A. An herbicide that inhibits the growing
point or meristem, such as an imidazolinone or a sulfonylurea. [0132] B.
Glyphosate (resistance conferred by mutant
5-enolpyruvylshikimate-3-phosphate synthase (EPSP) and aroA genes,
respectively) and other phosphono compounds such as glufosinate
(phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus
PAT bar genes), and pyridinoxy or phenoxy proprionic acids and
cyclohexones (ACCase inhibitor-encoding genes). Glyphosate resistance is
also imparted to plants that express a gene that encodes a glyphosate
oxido-reductase enzyme. In addition glyphosate resistance can be imparted
to plants by the over expression of genes encoding glyphosate
N-acetyltransferase. Nucleotide sequences of glutamine synthetase genes
which confer resistance to herbicides such as L-phosphinothricin are
known and can be used herein. The nucleotide sequence of a PAT gene is
also known and can be used. Exemplary of genes conferring resistance to
phenoxy proprionic acids and cyclohexones, such as sethoxydim and
haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes. [0133] C. An
herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+
genes) and a benzonitrile (nitrilase gene). The transformation of
Chlamydomonas with plasmids encoding mutant psbA genes are known and can
be used. [0134] D. Acetohydroxy acid synthase, which has been found to
make plants that express this enzyme resistant to multiple types of
herbicides. Other genes that confer tolerance to herbicides include a
gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast
NADPH-cytochrome P450 oxidoreductase, genes for glutathione reductase and
superoxide dismutase, and genes for various phosphotransferases. [0135]
E. Protoporphyrinogen oxidase (protox), which is necessary for the
production of chlorophyll. The protox enzyme serves as the target for a
variety of herbicidal compounds. These herbicides also inhibit growth of
all the different species of plants present, causing their total
destruction. 3. Genes that Confer or Contribute to a Value-Added Trait,
Such as: [0136] A. Modified fatty acid metabolism, for example, by
transforming a plant with an antisense gene of stearyl-ACP desaturase to
increase stearic acid content of the plant. [0137] B. Decreased phytate
content. Introduction of a phytase-encoding gene, such as Aspergillus
niger phytase gene, may enhance breakdown of phytate, adding more free
phosphate to the transformed plant. Alternatively, a gene could be
introduced that reduces phytate content. In maize for example, this could
be accomplished by cloning and then reintroducing DNA associated with the
single allele which is responsible for maize mutants characterized by low
levels of phytic acid. [0138] C. Modified carbohydrate composition
effected, for example, by transforming plants with a gene coding for an
enzyme that alters the branching pattern of starch, or, a gene altering
thioredoxin such as NTR and/or TRX and/or a gamma zein knock out or
mutant such as cs27 or TUSC27 or en27. Any known fatty acid modification
genes may also be used to affect starch content and/or composition
through the interrelationship of the starch and oil pathways. [0139] D.
Elevated oleic acid via FAD-2 gene modification and/or decreased
linolenic acid via FAD-3 gene modification. [0140] E. Altering conjugated
linolenic or linoleic acid content. Altering LEC1, AGP, Dek1, Superal1,
mi1ps, various Ipa genes such as Ipa1, Ipa3, hpt or hggt. [0141] F.
Altered antioxidant content or composition, such as alteration of
tocopherol or tocotrienols. In an embodiment, antioxidant levels may be
manipulated through alteration of a phytl prenyl transferase (ppt) or
through alteration of a homogentisate geranyl geranyl transferase (hggt).
[0142] G. Altered essential seed amino acids. 4. Genes that Control Male
Sterility [0143] There are several methods of conferring genetic male
sterility available and within the scope of the invention. As one
example, nuclear male sterility may be accomplished by identifying a gene
which is critical to male fertility, silencing this native gene which is
critical to male fertility, removing the native promoter from the
essential male fertility gene and replacing it with an inducible
promoter, inserting this genetically engineered gene back into the plant,
and thus creating a plant that is male sterile because the inducible
promoter is not "on," resulting in the male fertility gene not being
transcribed. Fertility is restored by inducing, or turning "on", the
promoter, which in turn allows the gene that confers male fertility to be
transcribed. Other possible examples include the tntroduction of a
deacetylase gene under the control of a tapetum-specific promoter and
with the application of the chemical N-Ac-PPT, the introduction of
various stamen-specific promoters, or the introduction of the barnase and
the barstar genes. 5. Genes that Create a Site for Site Specific DNA
Integration. [0144] This may include the introduction of FRT sites that
may be used in the FLP/FRT system and/or Lox sites that may be used in
the Cre/Loxp system. Other systems that may be used include the Gin
recombinase of phage Mu, the Pin recombinase of E. coli, and the R/RS
system of the pSR1 plasmid. 6. Genes that Affect Abiotic Stress
Resistance (Including but not Limited to flowering, pod and seed
development, enhancement of nitrogen utilization efficiency, altered
nitrogen responsiveness, drought resistance or tolerance, cold resistance
or tolerance, and salt resistance or tolerance) and increased yield under
stress. Genes and transcription factors that affect plant growth and
agronomic traits such as yield, flowering, plant growth and/or plant
structure, can be introduced or introgressed into plants.

Methods for Canola Transformation

[0145] Numerous methods for plant transformation have been developed,
including biological and physical plant transformation protocols. In
addition, expression vectors and in vitro culture methods for plant cell
or tissue transformation and regeneration of plants are available.

[0146] A. Agrobacterium-mediated Transformation--One method for
introducing an expression vector into plants is based on the natural
transformation system of Agrobacterium. A. tumefaciens and A. rhizogenes
are plant pathogenic soil bacteria which genetically transform plant
cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,
respectively, carry genes responsible for genetic transformation of the
plant. Agrobacterium vector systems and methods for
Agrobacterium-mediated gene transfer can be used in the present
invention.

[0147] B. Direct Gene Transfer--Several methods of plant transformation,
collectively referred to as direct gene transfer, have been developed as
an alternative to Agrobacterium-mediated transformation. A generally
applicable method of plant transformation is microprojectile-mediated
transformation wherein DNA is carried on the surface of microprojectiles
measuring 1 to 4 μm. The expression vector is introduced into plant
tissues with a ballistic device that accelerates the microprojectiles to
speeds of 300 to 600 m/s which is sufficient to penetrate plant cell
walls and membranes. Another method for physical delivery of DNA to
plants is sonication of target cells, which may be used herein.
Alternatively, liposome and spheroplast fusion may be used to introduce
expression vectors into plants. Direct uptake of DNA into protoplasts
using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine may
also be useful. Electroporation of protoplasts and whole cells and
tissues may also be utilized.

[0148] Following transformation of canola target tissues, expression of
the above-described selectable marker genes allows for preferential
selection of transformed cells, tissues and/or plants, using regeneration
and selection methods well known in the art.

[0149] The foregoing methods for transformation would typically be used
for producing a transgenic variety. The transgenic variety could then be
crossed with another (non-transformed or transformed) variety in order to
produce a new transgenic variety. Alternatively, a genetic trait which
has been engineered into a particular canola line using the foregoing
transformation techniques could be moved into another line using
traditional backcrossing techniques that are well known in the plant
breeding arts. For example, a backcrossing approach could be used to move
an engineered trait from a public, non-elite variety into an elite
variety, or from a variety containing a foreign gene in its genome into a
variety or varieties which do not contain that gene. As used herein,
"crossing" can refer to a simple X by Y cross, or the process of
backcrossing, depending on the context.

Genetic Marker Profile Through SSR and First Generation Progeny

[0150] In addition to phenotypic observations, a plant can also be
identified by its genotype. The genotype of a plant can be characterized
through a genetic marker profile which can identify plants of the same
variety or a related variety or be used to determine or validate a
pedigree. Genetic marker profiles can be obtained by techniques such as
Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified
Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction
(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized
Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms
(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to as
Microsatellites, and Single Nucleotide Polymorphisms (SNPs).

[0151] Particular markers used for these purposes are not limited to any
particular set of markers, but are envisioned to include any type of
marker and marker profile which provides a means of distinguishing
varieties. One method of comparison is to use only homozygous loci for
ND-662c.

[0152] In addition to being used for identification of canola line ND-662c
and plant parts and plant cells of line ND-662c, the genetic profile may
be used to identify a canola plant produced through the use of ND-662c or
to verify a pedigree for progeny plants produced through the use of
ND-662c. The genetic marker profile is also useful in breeding and
developing backcross conversions.

[0153] The present invention comprises a canola plant characterized by
molecular and physiological data obtained from the representative sample
of said variety deposited with the American Type Culture Collection
(ATCC). Further provided by the invention is a canola plant formed by the
combination of the disclosed canola plant or plant cell with another
canola plant or plant cell and comprising the homozygous alleles of the
variety.

[0154] Means of performing genetic marker profiles using SSR polymorphisms
are well known in the art. SSRs are genetic markers based on
polymorphisms in repeated nucleotide sequences, such as microsatellites.
A marker system based on SSRs can be highly informative in linkage
analysis relative to other marker systems in that multiple alleles may be
present. Another advantage of this type of marker is that, through use of
flanking primers, detection of SSRs can be achieved, for example, by the
polymerase chain reaction (PCR), thereby eliminating the need for
labor-intensive Southern hybridization. The PCR detection is done by use
of two oligonucleotide primers flanking the polymorphic segment of
repetitive DNA. Repeated cycles of heat denaturation of the DNA followed
by annealing of the primers to their complementary sequences at low
temperatures, and extension of the annealed primers with DNA polymerase,
comprise the major part of the methodology.

[0155] Following amplification, markers can be scored by electrophoresis
of the amplification products. Scoring of marker genotype is based on the
size of the amplified fragment, which may be measured by the number of
base pairs of the fragment. While variation in the primer used or in
laboratory procedures can affect the reported fragment size, relative
values should remain constant regardless of the specific primer or
laboratory used. When comparing varieties it is preferable if all SSR
profiles are performed in the same lab.

[0156] The SSR profile of canola plant ND-662c can be used to identify
plants comprising ND-662c as a parent, since such plants will comprise
the same homozygous alleles as ND-662c. Because the canola variety is
essentially homozygous at all relevant loci, most loci should have only
one type of allele present. In contrast, a genetic marker profile of an
F1 progeny should be the sum of those parents, e.g., if one parent
was homozygous for allele x at a particular locus, and the other parent
homozygous for allele y at that locus, then the F1 progeny will be
xy (heterozygous) at that locus. Subsequent generations of progeny
produced by selection and breeding are expected to be of genotype x
(homozygous), y (homozygous), or xy (heterozygous) for that locus
position. When the F1 plant is selfed or sibbed for successive
filial generations, the locus should be either x or y for that position.

[0157] In addition, plants and plant parts substantially benefiting from
the use of ND-662c in their development, such as ND-662c comprising a
backcross conversion, transgene, or genetic sterility factor, may be
identified by having a molecular marker profile with a high percent
identity to ND-662c. Such a percent identity might be 95%, 96%, 97%, 98%,
99%, 99.5% or 99.9% identical to ND-662c.

[0158] The SSR profile of ND-662c also can be used to identify essentially
derived varieties and other progeny varieties developed from the use of
ND-662c, as well as cells and other plant parts thereof. Progeny plants
and plant parts produced using ND-662c may be identified by having a
molecular marker profile of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
99.5% genetic contribution from canola variety, as measured by either
percent identity or percent similarity. Such progeny may be further
characterized as being within a pedigree distance of ND-662c, such as
within 1, 2, 3, 4 or 5 or less cross-pollinations to a canola plant other
than ND-662c or a plant that has ND-662c as a progenitor. Unique
molecular profiles may be identified with other molecular tools such as
SNPs and RFLPs.

[0159] While determining the SSR genetic marker profile of the plants
described supra, several unique SSR profiles may also be identified which
did not appear in either parent of such plant. Such unique SSR profiles
may arise during the breeding process from recombination or mutation. A
combination of several unique alleles provides a means of identifying a
plant variety, an F1 progeny produced from such variety, and progeny
produced from such variety.

Single-Gene Conversions

[0160] When the term "canola plant" is used in the context of the present
invention, this also includes any single gene conversions of that
variety. The term single gene converted plant as used herein refers to
those canola plants which are developed by backcrossing, wherein
essentially all of the desired morphological and physiological
characteristics of a variety are recovered in addition to the single gene
transferred into the variety via the backcrossing technique. Backcrossing
methods can be used with the present invention to improve or introduce a
characteristic into the variety. A hybrid progeny may be backcrossed to
the recurrent parent 1, 2, 3, 4, 5, 6, 7, 8 or more times as part of this
invention. The parental canola plant that contributes the gene for the
desired characteristic is termed the nonrecurrent or donor parent. This
terminology refers to the fact that the nonrecurrent parent is used one
time in the backcross protocol and therefore does not recur. The parental
canola plant to which the gene or genes from the nonrecurrent parent are
transferred is known as the recurrent parent as it is used for several
rounds in the backcrossing protocol. In a typical backcross protocol, the
original variety of interest (recurrent parent) is crossed to a second
variety (nonrecurrent parent) that carries the single gene of interest to
be transferred. The resulting progeny from this cross are then crossed
again to the recurrent parent and the process is repeated until a canola
plant is obtained wherein essentially all of the desired morphological
and physiological characteristics of the recurrent parent are recovered
in the converted plant, in addition to the single transferred gene from
the nonrecurrent parent.

[0161] The selection of a suitable recurrent parent is an important step
for a successful backcrossing procedure. The goal of a backcross protocol
is to alter or substitute a single trait or characteristic in the
original variety. To accomplish this, a single gene of the recurrent
variety is modified or substituted with the desired gene from the
nonrecurrent parent, while retaining essentially all of the rest of the
desired genetic, and therefore the desired physiological and
morphological, constitution of the original variety. The choice of the
particular nonrecurrent parent will depend on the purpose of the
backcross; one of the major purposes is to add some agronomically
important trait to the plant. The exact backcrossing protocol will depend
on the characteristic or trait being altered to determine an appropriate
testing protocol. Although backcrossing methods are simplified when the
characteristic being transferred is a dominant allele, a recessive allele
may also be transferred. In this instance it may be necessary to
introduce a test of the progeny to determine if the desired
characteristic has been successfully transferred.

[0162] Many single gene traits have been identified that are not regularly
selected for in the development of a new variety but that can be improved
by backcrossing techniques. Single gene traits may or may not be
transgenic; examples of these traits include but are not limited to, male
sterility, waxy starch, herbicide resistance, resistance for bacterial,
fungal, or viral disease, insect resistance, male fertility, enhanced
nutritional quality, industrial usage, yield stability and yield
enhancement. These genes are generally inherited through the nucleus.

Introduction of a New Trait or Locus into ND-662c

[0163] Line ND-662c represents a new base genetic variety into which a new
locus or trait may be introgressed. Direct transformation and
backcrossing represent two methods that can be used to accomplish such an
introgression. The term backcross conversion and single locus conversion
are used interchangeably to designate the product of a backcrossing
program.

Backcross Conversions of ND-662c

[0164] A backcross conversion of ND-662c may occur when DNA sequences are
introduced through backcrossing with ND-662c utilized as the recurrent
parent. Both naturally occurring and transgenic DNA sequences may be
introduced through backcrossing techniques. Molecular marker assisted
breeding or selection may be utilized to reduce the number of backcrosses
necessary to achieve the backcross conversion.

[0165] The complexity of the backcross conversion method depends on the
type of trait being transferred (single genes or closely linked genes as
vs. unlinked genes), the level of expression of the trait, the type of
inheritance (cytoplasmic or nuclear) and the types of parents included in
the cross. It is understood by those of ordinary skill in the art that
for single gene traits that are relatively easy to classify, the
backcross method is effective and relatively easy to manage. Desired
traits that may be transferred through backcross conversion include, but
are not limited to, sterility (nuclear and cytoplasmic), fertility
restoration, nutritional enhancements, drought tolerance, nitrogen
utilization, altered fatty acid profile, altered seed amino acid levels,
altered seed oil levels, low phytate, industrial enhancements, disease
resistance (bacterial, fungal or viral), insect resistance and herbicide
resistance. In addition, an introgression site itself, such as an FRT
site, Lox site or other site specific integration site, may be inserted
by backcrossing and utilized for direct insertion of one or more genes of
interest into a specific plant variety. In some embodiments of the
invention, the number of loci that may be backcrossed into ND-662c is at
least 1, 2, 3, 4, or 5 and/or no more than 6, 5, 4, 3, or 2. A single
locus may contain several transgenes, such as a transgene for disease
resistance that, in the same expression vector, also contains a transgene
for herbicide resistance. The gene for herbicide resistance may be used
as a selectable marker and/or as a phenotypic trait. A single locus
conversion of site specific integration system allows for the integration
of multiple genes at the converted loci.

[0166] The backcross conversion may result from either the transfer of a
dominant allele or a recessive allele. Selection of progeny containing
the trait of interest is accomplished by direct selection for a trait
associated with a dominant allele. Transgenes transferred via
backcrossing typically function as a dominant single gene trait and are
relatively easy to classify. Selection of progeny for a trait that is
transferred via a recessive allele requires growing and selfing the first
backcross generation to determine which plants carry the recessive
alleles. Recessive traits may require additional progeny testing in
successive backcross generations to determine the presence of the locus
of interest. The last backcross generation is usually selfed to give pure
breeding progeny for the gene(s) being transferred, although a backcross
conversion with a stably introgressed trait may also be maintained by
further backcrossing to the recurrent parent with selection for the
converted trait.

[0167] Along with selection for the trait of interest, progeny are
selected for the phenotype of the recurrent parent. The backcross is a
form of inbreeding, and the features of the recurrent parent are
automatically recovered after successive backcrosses. As noted above, the
number of backcrosses necessary can be reduced with the use of molecular
markers. Other factors, such as a genetically similar donor parent, may
also reduce the number of backcrosses necessary. As noted, backcrossing
is easiest for simply inherited, dominant and easily recognized traits.

[0168] One process for adding or modifying a trait or locus in canola line
ND-662c comprises crossing ND-662c plants grown from ND-662c seed with
plants of another canola variety that comprise the desired trait or
locus, selecting Ft progeny plants that comprise the desired trait
or locus to produce selected F1 progeny plants, crossing the
selected progeny plants with the ND-662c plants to produce backcross
progeny plants, selecting for backcross progeny plants that have the
desired trait or locus and the morphological characteristics of canola
line ND-662c to produce selected backcross progeny plants; and
backcrossing to ND-662c three or more times in succession to produce
selected fourth or higher backcross progeny plants that comprise said
trait or locus. The modified ND-662c may be further characterized as
having essentially all of the physiological and morphological
characteristics of canola line ND-662c listed in Table 1 and/or may be
characterized by percent similarity or identity to ND-662c as determined
by SSR markers. The above method may be utilized with fewer backcrosses
in appropriate situations, such as when the donor parent is highly
related or markers are used in the selection step. Desired traits that
may be used include those nucleic acids known in the art, some of which
are listed herein, that will affect traits through nucleic acid
expression or inhibition. Desired loci include the introgression of FRT,
Lox and other sites for site specific integration, which may also affect
a desired trait if a functional nucleic acid is inserted at the
integration site.

[0169] In addition, the above process and other similar processes
described herein may be used to produce first generation progeny canola
seed by adding a step at the end of the process that comprises crossing
ND-662c with the introgressed trait or locus with a different canola
plant and harvesting the resultant first generation progeny canola seed.

Tissue Culture of Canola

[0170] Further production of the ND-662c line can occur by tissue culture
and regeneration. Culture of various tissues of canola and regeneration
of plants therefrom is known and widely published. Thus, another aspect
of this invention is to provide cells which upon growth and
differentiation produce canola plants having the physiological and
morphological characteristics of canola line ND-662c.

[0171] As used herein, the term "tissue culture" indicates a composition
comprising isolated cells of the same or a different type or a collection
of such cells organized into parts of a plant. Exemplary types of tissue
cultures are protoplasts, calli, plant clumps, and plant cells that can
generate tissue culture that are intact in plants or parts of plants,
such as embryos, pollen, flowers, seeds, pods, leaves, stems, roots, root
tips, anthers, pistils and the like. Means for preparing and maintaining
plant tissue culture are well known in the art. Tissue culture comprising
organs can be used in the present invention to produce regenerated
plants.

Using ND-662c to Develop Other Canola Varieties

[0172] Canola varieties such as ND-662c are typically developed for use in
seed and grain production. However, canola varieties such as ND-662c also
provide a source of breeding material that may be used to develop new
canola varieties. Plant breeding techniques known in the art that can be
used in a canola plant breeding program according to the invention
include, but are not limited to, recurrent selection, mass selection,
bulk selection, mass selection, backcrossing, pedigree breeding, open
pollination breeding, restriction fragment length polymorphism enhanced
selection, genetic marker enhanced selection, making double haploids, and
transformation. Often combinations of these techniques are used. The
development of canola varieties in a plant breeding program requires, in
general, the development and evaluation of homozygous varieties.

Additional Breeding Methods

[0173] This invention is directed to methods for producing a canola plant
by crossing a first parent canola plant with a second parent canola plant
wherein either the first or second parent canola plant is line ND-662c.
The other parent may be any other canola plant, such as a canola plant
that is part of a synthetic or natural population. Any such methods using
canola line ND-662c are part of this invention: selfing, sibbing,
backcrosses, mass selection, pedigree breeding, bulk selection, hybrid
production, crosses to populations, and the like. These methods are well
known in the art and some of the more commonly used breeding methods are
described below.

[0174] The following describes breeding methods that may be used with
canola line ND-662c in the development of further canola plants. One such
embodiment is a method for developing a line ND-662c progeny canola plant
in a canola plant breeding program comprising: obtaining the canola
plant, or a part thereof, of line ND-662c utilizing said plant or plant
part as a source of breeding material and selecting a canola line ND-662c
progeny plant with molecular markers in common with line ND-662c and/or
with morphological and/or physiological characteristics selected from the
characteristics listed in Table 1. Breeding steps that may be used in the
canola plant breeding program include pedigree breeding, backcrossing,
mutation breeding, and recurrent selection. In conjunction with these
steps, techniques such as RFLP-enhanced selection, genetic marker
enhanced selection (for example SSR markers) and the making of double
haploids may be utilized.

[0175] Another method involves producing a population of canola line
ND-662c progeny canola plants, comprising crossing line ND-662c with
another canola plant, thereby producing a population of canola plants,
which, on average, derive 50% of their alleles from canola line ND-662c.
A plant of this population may be selected and repeatedly selfed or
sibbed with a canola line resulting from these successive filial
generations. One embodiment of this invention is the canola line produced
by this method and that has obtained at least 50% of its alleles from
canola line ND-662c.

[0176] One of ordinary skill in the art of plant breeding would know how
to evaluate the traits of two plant varieties to determine if there is no
significant difference between the two traits expressed by those
varieties. Thus the invention includes canola line ND-662c progeny canola
plants comprising a combination of at least two line ND-662c traits
selected from the group consisting of those listed in Table 1 or the line
ND-662c combination of traits listed in the Summary of the Invention, so
that said progeny canola plant is not significantly different for said
traits than canola line ND-662c as determined at the 5% significance
level when grown in the same environmental conditions. Using techniques
described herein, molecular markers may be used to identify said progeny
plant as a canola line ND-662c progeny plant. Mean trait values may be
used to determine whether trait differences are significant, and
preferably the traits are measured on plants grown under the same
environmental conditions. Once such a variety is developed its value is
substantial since it is important to advance the germplasm base as a
whole in order to maintain or improve traits such as yield, disease
resistance, pest resistance, and plant performance in extreme
environmental conditions.

[0177] Progeny of canola line ND-662c may also be characterized through
their filial relationship with canola line ND-662c, as for example, being
within a certain number of breeding crosses of canola line ND-662c. A
breeding cross is a cross made to introduce new genetics into the
progeny, and is distinguished from a cross, such as a self or a sib
cross, made to select among existing genetic alleles. The lower the
number of breeding crosses in the pedigree, the closer the relationship
between canola line ND-662c and its progeny. For example, progeny
produced by the methods described herein may be within 1, 2, 3, 4 or 5
breeding crosses of canola line ND-662c.

[0178] As used herein, the term "plant" includes plant cells, plant
protoplasts, plant cell tissue cultures from which canola plants can be
regenerated, plant calli, plant clumps, and plant cells that are intact
in plants or parts of plants, such as embryos, pollen, ovules, flowers,
pods, leaves, roots, root tips, anthers, cotyledons, hypocotyls,
meristematic cells, stems, pistils, petiole, and the like.

Pedigree Breeding

[0179] Pedigree breeding starts with the crossing of two genotypes, such
as ND-662c and another canola variety having one or more desirable
characteristics that is lacking or which complements ND-662c. If the two
original parents do not provide all the desired characteristics, other
sources can be included in the breeding population. In the pedigree
method, superior plants are selfed and selected in successive filial
generations. In the succeeding filial generations the heterozygous
condition gives way to homogeneous varieties as a result of
self-pollination and selection. Typically in the pedigree method of
breeding, five or more successive filial generations of selfing and
selection is practiced: F1 to F2; F2 to F3; F3
to F4; F4 to F5, etc. After a sufficient amount of
inbreeding, successive filial generations will serve to increase seed of
the developed variety. Preferably, the developed variety comprises
homozygous alleles at about 95% or more of its loci.

[0180] In addition to being used to create a backcross conversion,
backcrossing can also be used in combination with pedigree breeding. As
discussed previously, backcrossing can be used to transfer one or more
specifically desirable traits from one variety, the donor parent, to a
developed variety called the recurrent parent, which has overall good
agronomic characteristics yet lacks that desirable trait or traits.
However, the same procedure can be used to move the progeny toward the
genotype of the recurrent parent but at the same time retain many
components of the non-recurrent parent by stopping the backcrossing at an
early stage and proceeding with selfing and selection. For example, a
canola variety may be crossed with another variety to produce a first
generation progeny plant. The first generation progeny plant may then be
backcrossed to one of its parent varieties to create a BC1 or BC2.
Progeny are selfed and selected so that the newly developed variety has
many of the attributes of the recurrent parent and yet several of the
desired attributes of the non-recurrent parent. This approach leverages
the value and strengths of the recurrent parent for use in new canola
varieties.

[0181] Therefore, an embodiment of this invention is a method of making a
backcross conversion of canola line ND-662c, comprising the steps of
crossing a plant of canola line ND-662c with a donor plant comprising a
desired trait, selecting an F1 progeny plant comprising the desired
trait, and backcrossing the selected F1 progeny plant to a plant of
canola line ND-662c. This method may further comprise the step of
obtaining a molecular marker profile of canola line ND-662c and using the
molecular marker profile to select for a progeny plant with the desired
trait and the molecular marker profile of ND-662c. In one embodiment the
desired trait is a mutant gene or transgene present in the donor parent.

Recurrent Selection and Mass Selection

[0182] Recurrent selection is a method used in a plant breeding program to
improve a population of plants. ND-662c is suitable for use in a
recurrent selection program. The method entails individual plants cross
pollinating with each other to form progeny. The progeny are grown and
the superior progeny selected by any number of selection methods, which
include individual plant, half-sib progeny, full-sib progeny and selfed
progeny. The selected progeny are cross pollinated with each other to
form progeny for another population. This population is planted and again
superior plants are selected to cross pollinate with each other.
Recurrent selection is a cyclical process and therefore can be repeated
as many times as desired. The objective of recurrent selection is to
improve the traits of a population. The improved population can then be
used as a source of breeding material to obtain new varieties for
commercial or breeding use, including the production of a synthetic line.
A synthetic line is the resultant progeny formed by the intercrossing of
several selected varieties.

[0183] Mass selection is a useful technique when used in conjunction with
molecular marker enhanced selection. In mass selection seeds from
individuals are selected based on phenotype or genotype. These selected
seeds are then bulked and used to grow the next generation. Bulk
selection requires growing a population of plants in a bulk plot,
allowing the plants to self-pollinate, harvesting the seed in bulk and
then using a sample of the seed harvested in bulk to plant the next
generation. Also, instead of self pollination, directed pollination could
be used as part of the breeding program.

Mutation Breeding

[0184] Mutation breeding is another method of introducing new traits into
canola line ND-662c. Mutations that occur spontaneously or are
artificially induced can be useful sources of variability for a plant
breeder. The goal of artificial mutagenesis is to increase the rate of
mutation for a desired characteristic. Mutation rates can be increased by
many different means including temperature, long-term seed storage,
tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.
cobalt 60 or cesium 137), neutrons, (product of nuclear fission by
uranium 235 in an atomic reactor), Beta radiation (emitted from
radioisotopes such as phosphorus 32 or carbon 14), or ultraviolet
radiation (preferably from 2500 to 2900 nm), or chemical mutagens (such
as base analogues (5-bromo-uracil), related compounds (8-ethoxy
caffeine), antibiotics (streptonigrin), alkylating agents (sulfur
mustards, nitrogen mustards, epoxides, ethylenamines, sulfates,
sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, or
acridines. Once a desired trait is observed through mutagenesis the trait
may then be incorporated into existing germplasm by traditional breeding
techniques. In addition, mutations created in other canola plants may be
used to produce a backcross conversion of canola line ND-662c that
comprises such mutation.

Breeding with Molecular Markers

[0185] Molecular markers, which include markers identified through the use
of techniques such as Isozyme Electrophoresis, Restriction Fragment
Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs
(RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA
Amplification Fingerprinting (DAF), Sequence Characterized Amplified
Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), Simple
Sequence Repeats (SSRs) and Single Nucleotide Polymorphisms (SNPs), may
be used in plant breeding methods utilizing canola line ND-662c. One use
of molecular markers is Quantitative Trait Loci (QTL) mapping. QTL
mapping is the use of markers, which are known to be closely linked to
alleles that have measurable effects on a quantitative trait. Selection
in the breeding process is based upon the accumulation of markers linked
to the positive effecting alleles and/or the elimination of the markers
linked to the negative effecting alleles from the plant's genome.

[0186] Molecular markers can also be used during the breeding process for
the selection of qualitative traits. For example, markers closely linked
to alleles or markers containing sequences within the actual alleles of
interest can be used to select plants that contain the alleles of
interest during a backcrossing breeding program. The markers can also be
used to select for the genome of the recurrent parent and against the
genome of the donor parent. Using this procedure can minimize the amount
of genome from the donor parent that remains in the selected plants. It
can also be used to reduce the number of crosses back to the recurrent
parent needed in a backcrossing program. The use of molecular markers in
the selection process is often called genetic marker enhanced selection.
Molecular markers may also be used to identify and exclude certain
sources of germplasm as parental varieties or ancestors of a plant by
providing a means of tracking genetic profiles through crosses.

Production of Double Haploids

[0187] The production of double haploids can also be used for the
development of plants with a homozygous phenotype in the breeding
program. For example, a canola plant for which canola line ND-662c is a
parent can be used to produce double haploid plants. Double haploids are
produced by the doubling of a set of chromosomes (1 N) from a
heterozygous plant to produce a completely homozygous individual. This
can be advantageous because the process omits the generations of selfing
needed to obtain a homozygous plant from a heterozygous source.

[0188] Haploid induction systems have been developed for various plants to
produce haploid tissues, plants and seeds. Thus, an embodiment of this
invention is a process for making a substantially homozygous ND-662c
progeny plant by producing or obtaining a seed from the cross of ND-662c
and another canola plant and applying double haploid methods to the
F1 seed or F1 plant or to any successive filial generation.
Based on studies in maize and currently being conducted in canola, such
methods would decrease the number of generations required to produce a
variety with similar genetics or characteristics to ND-662c.

[0189] In particular, a process of making seed retaining the molecular
marker profile of canola line ND-662c is contemplated, such process
comprising obtaining or producing F1 seed for which canola line
ND-662c is a parent, inducing doubled haploids to create progeny without
the occurrence of meiotic segregation, obtaining the molecular marker
profile of canola line ND-662c, and selecting progeny that retain the
molecular marker profile of ND-662c.

[0190] A pollination control system and effective transfer of pollen from
one parent to the other offers improved plant breeding and an effective
method for producing hybrid canola seed and plants. For example, the
ogura cytoplasmic male sterility (cms) system, developed via protoplast
fusion between radish (Raphanus sativus) and rapeseed (Brassica napus) is
one of the most frequently used methods of hybrid production. It provides
stable expression of the male sterility trait and an effective nuclear
restorer gene.

[0191] In developing improved new Brassica hybrid varieties, breeders use
self-incompatible (SI), cytoplasmic male sterile (CMS) and nuclear male
sterile (NMS) Brassica plants as the female parent. In using these
plants, breeders are attempting to improve the efficiency of seed
production and the quality of the F1 hybrids and to reduce the
breeding costs. When hybridization is conducted without using SI, CMS or
NMS plants, it is more difficult to obtain and isolate the desired traits
in the progeny (F1 generation) because the parents are capable of
undergoing both cross-pollination and self-pollination. If one of the
parents is a SI, CMS or NMS plant that is incapable of producing pollen,
only cross pollination will occur. By eliminating the pollen of one
parental variety in a cross, a plant breeder is assured of obtaining
hybrid seed of uniform quality, provided that the parents are of uniform
quality and the breeder conducts a single cross.

[0192] In one instance, production of F1 hybrids includes crossing a
CMS Brassica female parent, with a pollen producing male Brassica parent.
To reproduce effectively, however, the male parent of the F1 hybrid
must have a fertility restorer gene (Rf gene). The presence of an Rf gene
means that the F1 generation will not be completely or partially
sterile, so that either self-pollination or cross pollination may occur.
Self pollination of the F1 generation to produce several subsequent
generations is important to ensure that a desired trait is heritable and
stable and that a new variety has been isolated.

[0193] An example of a Brassica plant which is cytoplasmic male sterile
and used for breeding is ogura (OGU) cytoplasmic male sterile. A
fertility restorer for ogura cytoplasmic male sterile plants has been
transferred from Raphanus sativus (radish) to Brassica. The restorer gene
is Rf1, originating from radish. Improved versions of this restorer have
been developed as well. Other sources and refinements of CMS sterility in
canola include the Polima cytoplasmic male sterile plant.

[0194] Further, as a result of the advances in sterility systems, lines
are developed that can be used as an open pollinated variety and/or as a
sterile inbred (female) used in the production of F1 hybrid seed. In
the latter case, favorable combining ability with a restorer (male) would
be desirable. The resulting hybrid seed would then be sold to the grower
for planting.

[0195] The development of a canola hybrid in a canola plant breeding
program involves three steps: (1) the selection of plants from various
germplasm pools for initial breeding crosses; (2) the selfing of the
selected plants from the breeding crosses for several generations to
produce a series of inbred lines, which, although different from each
other, breed true and are highly uniform; and (3) crossing the selected
inbred lines with different inbred lines to produce the hybrids. During
the inbreeding process in canola, the vigor of the lines decreases. Vigor
is restored when two different inbred lines are crossed to produce the
hybrid. An important consequence of the homozygosity and homogeneity of
the inbred lines is that the hybrid between a defined pair of inbreds
will always be the same. Once the inbreds that give a superior hybrid
have been identified, the hybrid seed can be reproduced indefinitely as
long as the homogeneity of the inbred parents is maintained.

[0196] Combining ability of a line, as well as the performance of the line
per se, is a factor in the selection of improved canola lines that may be
used as inbreds. Combining ability refers to a line's contribution as a
parent when crossed with other lines to form hybrids. The hybrids formed
for the purpose of selecting superior lines are designated test crosses.
One way of measuring combining ability is by using breeding values.
Breeding values are based on the overall mean of a number of test
crosses. This mean is then adjusted to remove environmental effects and
it is adjusted for known genetic relationships among the lines.

[0197] Hybrid seed production requires inactivation of pollen produced by
the female parent. Incomplete inactivation of the pollen provides the
potential for self-pollination. This inadvertently self-pollinated seed
may be unintentionally harvested and packaged with hybrid seed.
Similarly, because the male parent is grown next to the female parent in
the field there is also the potential that the male selfed seed could be
unintentionally harvested and packaged with the hybrid seed. Once the
seed from the hybrid bag is planted, it is possible to identify and
select these self-pollinated plants. These self-pollinated plants will be
genetically equivalent to one of the inbred lines used to produce the
hybrid. Though the possibility of inbreds being included in hybrid seed
bags exists, the occurrence is rare because much care is taken to avoid
such inclusions. These self-pollinated plants can be identified and
selected by one skilled in the art, either through visual or molecular
methods.

[0198] Brassica napus canola plants, absent the use of sterility systems,
are recognized to commonly be self-fertile with approximately 70 to 90
percent of the seed normally forming as the result of self-pollination.
The percentage of cross pollination may be further enhanced when
populations of recognized insect pollinators at a given growing site are
greater. Thus open pollination is often used in commercial canola
production and canola line ND-662c may be open pollinated as part of the
invention.

INDUSTRIAL USES

[0199] Currently Brassica napus canola is recognized as an increasingly
important oilseed crop and a source of meal in many parts of the world.
The oil as removed from the seeds commonly contains a lesser
concentration of endogenously formed saturated fatty acids than other
vegetable oils and is well suited for use in the production of salad oil
or other food products or in cooking or frying applications. The oil also
finds utility in industrial applications. Additionally, the meal
component of the seeds can be used as a nutritious protein concentrate
for livestock.

[0200] Canola oil has the lowest level of saturated fatty acids of all
vegetable oils. "Canola" refers to rapeseed (Brassica) which has a erucic
acid (C22:1) content of at most 2 percent by weight based on the
total fatty acid content of a seed, and which produces, after crushing,
an air-dried meal containing less than 30 micromoles (μmol) per gram
of defatted (oil-free) meal. These types of rapeseed are distinguished by
their edibility in comparison to more traditional varieties of the
species.

[0201] Canola line ND-662c can be used in the production of an edible
vegetable oil or other food products in accordance with known techniques.
The solid meal component derived from seeds can be used as a nutritious
livestock feed. Parts of the plant not used for human or animal food can
be used for biofuel.

[0203] Compared to the two commercial canola lines DKL38-25 and IS 71-45,
the values presented in Table 2 indicate that seed of canola variety
ND-662c of the present invention has a different oil quality profile that
is still within a normal range for canola. While its oil quality profile
is in the normal range, it is the higher oil yield of ND-662c (see Table
1) that is the more distinguishing characteristic of its oil makeup.

Deposit Information

[0204] A deposit of the proprietary canola line designated ND-662c,
disclosed above and recited in the appended claims, has been made with
the American Type Culture Collection (ATCC), University Boulevard,
Manassas, Va. 20110. The date of deposit was Jan. 15, 2011. All
restrictions upon the deposit have been irrevocably removed, and the
deposit is intended to meet all of the requirements of 37 C.F.R.
1.801-1.809. The ATCC accession number is PTA-11594. The deposit will be
maintained in the depository for a period of 30 years, or 5 years after
the last request, or for the effective life of the patent, whichever is
longer, and will be replaced as necessary during that period.

[0205] While a number of exemplary aspects and embodiments have been
discussed above, those of skill in the art will recognize certain
modifications, permutations, additions and sub-combinations thereof. It
is therefore intended that the following appended claims and claims
hereafter introduced are interpreted to include all such modifications,
permutations, additions and sub-combinations as are within their true
spirit and scope.